Overview of FGF/FGFR Signaling

The FGF/FGFR signaling system comprises FGFs (ligands) and FGFRs (receptors), along with essential regulatory components, including the co‐receptors Klotho (for endocrine FGFs), extracellular FGF binding proteins (FGFBPs), and heparan sulfate proteoglycans (HSPGs), which collectively govern FGF presentation, FGFR activation, signal transduction, and specificity (Figure 1).

FIGURE 1: Classification and functional signaling characteristics of the FGF family. Fibroblast growth factors (FGFs) can be divided into three major functional categories according to their structural features, modes of action, and receptor preferences: endocrine FGFs (represented by the FGF19 subfamily), paracrine FGFs (including the FGF1, FGF4, FGF7, FGF8, and FGF9 subfamilies), and intracellular FGFs (iFGFs, viz., the FGF11 subfamily). Endocrine FGFs assemble with FGFR and the specific Klotho (KLB) co‐receptor to form a functional signaling complex. Paracrine FGFs exert their biological functions by forming a signaling complex with FGFR, utilizing cell surface heparan sulfate proteoglycans (HSPGs) as co‐receptors. In contrast, intracellular FGFs (FGF11 subfamily) function independently of FGFR activation and directly act intracellularly to regulate neuronal excitability, MAPK, and other downstream signaling pathways, thereby mediating unique biological effects. Different FGF subfamilies also exhibit differential FGFR binding preferences. Created with Biorender.com.

The Components of FGF/FGFR Signaling

FGF Family

The human FGF family comprises 22 members, which are classified into five paracrine subfamilies (FGF1, FGF4, FGF7, FGF8, and FGF9), one endocrine subfamily (FGF19), and one intracellular subfamily (FGF11) based on sequence homology, phylogenetic relationships, and secretion modes [11]. This functional classification closely correlates with their distinct mechanisms of receptor engagement and signaling. Paracrine and endocrine FGFs activate FGFRs on the cell membrane to execute their functions, whereas intracellular subfamily FGF11 (also known as intracellular homologous factor) does not bind to FGFRs. Instead, it functions autonomously within the cytoplasm and nucleus, where it modulates ion channel activity. Specifically, FGF11 acts directly within the cell, mainly regulating intracellular voltage‐gated sodium channels and other proteins, thereby participating in regulation of neuronal excitation and cardiac electrical activity [12, 13].

All FGF members are composed of 150–300 amino acids (AAs) and share a conserved core domain of approximately 125 AAs. This conserved core domain adopts a canonical β‐trefoil fold formed by 12 antiparallel β‐strands, which constitutes the key region responsible for receptor binding and exerts its functions [14, 15]. Moreover, this domain contains typical heparan sulfate (HS) binding sites that enable paracrine FGFs to anchor to HSPGs on the cell surface, thereby facilitating the formation of a stable FGF–FGFR–HS ternary complex and the subsequent initiation of intracellular signal transduction [16]. Nevertheless, marked structural and functional divergence exists across FGF subfamilies. Endocrine FGFs lack the β11 strand, leading to an atypical β‐trefoil fold that impairs high‐affinity heparin binding. Consequently, they require the co‐receptor Klotho to bind with FGFR and trigger downstream signal transduction [12]. For instance, endocrine FGFs, including FGF19, FGF21, and FGF23, are secreted into the circulation and act on distal target organs co‐expressing both FGFRs and Klotho co‐receptors [17]. Moreover, the N‐terminal and C‐terminal regions of FGF family members exhibit pronounced sequence divergence, which confer a fundamental molecular determinant of FGF functional diversity [18].

Fibroblast Growth Factor Receptors

As the specific receptor of FGF ligands, FGFRs are a typical class of receptor tyrosine kinases (RTKs) that consist of four highly homologous receptor subtypes (FGFR1–FGFR4) [19]. As single‐pass transmembrane proteins, each FGFR subtype is composed of approximately 800 AA residues [11] and exhibits a conserved domain architecture that includes three immunoglobulin‐like extracellular domains (D1–D3), a hydrophobic transmembrane domain (TMD), and a cytoplasmic tyrosine kinase domain with catalytic activity [20, 21]. Among these domains, the D1 domain, in collaboration with the adjacent acidic box, mediates conformational autoinhibition, thereby effectively preventing receptor dimerization and spontaneous phosphorylation in the absence of ligands. Meanwhile, the D2 and D3 domains collectively form the core ligand‐binding region, in which sequence variations and spatial arrangement directly determine the recognition specificity and binding affinity for distinct FGF ligands [22, 23].

Alternative splicing of FGFR transcripts can precisely regulate the specificity of FGFR signaling. Notably, alternative splicing of the D3 domain in FGFR1–FGFR3 generates two distinct isoforms, designated as IIIb and IIIc [24], and this splicing event exhibits pronounced tissue specificity. Generally, epithelial cells predominantly express the FGFR‐IIIb subtype, which displays high affinity for mesenchymal cell‐derived FGF2 and FGF7. Conversely, mesenchymal cells primarily express the FGFR‐IIIc subtype, which shows high affinity for epithelial cell‐derived FGF8 [25].

In contrast, FGFR4 does not undergo alternative splicing to produce distinct isoforms, and no significant differences in FGF binding affinity have been observed between its long and short variants [26]. Collectively, this finely tuned splicing regulation of FGFRs, together with the tissue‐specific patterns of FGF–FGFR, constitutes a key mechanism for orchestrating the activation of the FGF/FGFR signaling and maintaining normal tissue homeostasis.

Klothos and HSPG

The precision of FGF/FGFR signaling depends on the fine‐tuned regulation of co‐receptor and binding proteins [27]. These accessory molecules enhance ligand–receptor affinity through complex structural mechanisms, modulate the transmission of FGF/FGFR signaling, and ensure the activation of downstream signaling pathways with high binding strength. The two most important types of accessory molecules are the Klotho proteins and HSPGs.

Klotho proteins serve as essential co‐receptors mediating the biological effects of endocrine FGFs (FGF19, FGF21, and FGF23) and primarily comprise two subtypes: α‐Klotho and β‐Klotho [28, 29]. Both are single‐pass transmembrane proteins, and their extracellular regions contain two glycosidase homology domains, designated Klotho homology domain 1 (KL1) and Klotho homology domain 2 (KL2). Throughout evolution, although these two domains have lost their original enzymatic catalytic activity, they have acquired new protein interaction interfaces, thereby gaining the function of co‐receptors [30]. In contrast to paracrine FGFs, which rely on electrostatic interactions with HSPGs for stabilization, endocrine FGFs exhibit markedly reduced heparin‐binding affinity due to conformational alterations in the β11 strand of their core domain. Consequently, endocrine FGFs require Klotho proteins as molecular scaffolds to achieve receptor dimerization [29, 31].

Cryo‐electron microscopy structural analysis has revealed the fine architecture of the FGF–FGFR–Klotho ternary complex. The extracellular domain of the Klotho protein, via its KL2 domain, simultaneously engages the C‐terminus of the FGF ligand and the D3 domain of FGFR, thereby forcibly bringing the receptor and ligand under conditions of low heparin affinity and inducing the formation of a stable signaling complex [32, 33]. This binding mode exhibits extremely high selectivity. For instance, α‐Klotho specifically assists FGF23 in activating FGFR1c to regulate phosphorus metabolism, whereas β‐Klotho mainly assists FGF19 and FGF21 in activating FGFR4 and FGFR1c, respectively, participating in the regulation of BA and lipid metabolism [29, 34]. In addition to functioning as a transmembrane co‐receptor, Klotho family proteins also exhibit remarkable molecular versatility. Their extracellular domains can be cleaved and shed by proteases, such as ADAM10/17, and enter the bloodstream as soluble Klotho (sKlotho). sKlotho is a biologically active form that participates in several critical physiological functions, such as anti‐oxidative stress, maintaining ion channel homeostasis, and delaying cellular senescence [28].

HSPGs are a class of large composite molecules composed of a core protein and covalently linked to HS‐type glycosaminoglycan (GAG) chains [35]. HSPGs are widely distributed on the cell membrane surface and within the extracellular matrix (ECM) of all animal cells. On the basis of their localization and structure characteristics, they can be categorized into three types: membrane HSPGs, the secreted ECM HSPGs, and the secretory vesicle proteoglycan [36, 37]. The molecular weight of HSPG core proteins varies considerably, ranging from approximately 37 to 400 kDa. The binding of HSPGs to their ligands mainly depends on the sulfated HS chains they carry [38]. The sulfation modification of HS chains plays a key role in stabilizing the FGF–FGFR–HS ternary complex and in regulating FGF signal transduction [39, 40]. X‐ray crystallography analysis has revealed the structural characteristics of the ternary complex, confirming that HS acts as a molecular bridge between FGF and the D2 domain of FGFR by simultaneously binding to both. This interaction effectively induces the formation of a stable 2:2:2 dimeric complex and enhances the binding affinity between FGF and FGFR [41].

FGF Binding Proteins

FGFBPs constitute a class of secreted carrier proteins that can bind to specific FGFs, such as FGF1, FGF2, FGF7, and FGF10, with high specificity in a non‐covalent manner, thereby mobilizing locally acting paracrine FGFs from their extracellular storage [42, 43]. The FGFBP family comprises FGFBP1 (also known as heparin‐binding protein, HBP17), FGFBP2, and FGFBP3 (also known as killer‐specific secretory protein, Ksp37). These proteins typically consist of approximately 250 AAs and are secreted carrier proteins with a molecular weight of approximately 25 kDa [42]. They possess two characteristic functional domains: The N‐terminal domain interacts with heparin chains in the ECM, whereas the C‐terminal domain specifically recognizes the β‐tricyclic domain of FGFs [43, 44]. Under physiological homeostasis, the expression levels of FGFBP remain extremely low, and most FGFs are located in the extracelluar matrix. Upon exposure to tissue injury, inflammation, or malignant transformation, FGFBP expression is markedly upregulated. By taking advantage of their higher affinity than the ECM, FGFBPs competitively bind to FGF, forming FGF‐FGFBP complexes that enable FGFs to precisely engage FGFR binding pockets on adjacent or distant cell membranes [44, 45]. For instance, FGFBP1 binds to FGF5 through liquid–liquid phase separation (LLPS), thereby promoting liver repair [46]. Besides, the FGFBP3 protein interacts with endocrine FGFs through its C‐terminus, and thus potentiates the endocrine FGF/FGFR signaling [47].

The Downstream Signaling Cascades of FGF/FGFR Signaling

When FGF ligands form a ternary symmetrical complex with FGFR receptors and co‐factors such as HSPG or Klotho proteins, the conformation of the RTK domain changes and undergoes autophosphorylation, which further activates a series of downstream signaling cascades, such as RAS/MAPK, PI3K/AKT, PLCγ/PKC, and STAT pathways (Figure 2).

FIGURE 2: The FGF/FGFR signaling pathway. Following ligand binding and co‐receptor complex assembly, the tyrosine kinase domain of fibroblast growth factor receptor (FGFR) is activated, initiating a series of intracellular signaling cascades. Major downstream signaling axes include the RAS–MAPK, PI3K–AKT, JAK–STAT, and PLCγ‐Ca2+ pathways. These cascades ultimately regulate the transcription of target genes and thereby modulate fundamental cellular biological processes such as cell proliferation, differentiation, and survival. Paracrine FGF signaling depends on HSPG co‐receptors, whereas endocrine FGF signaling requires Klotho (KLB) for functional signaling complex formation. The entire signaling pathway is tightly and finely regulated via negative feedback by SPRY, SEF, and FGFRL1. Distinctively, intracellular FGFs (iFGFs) function in an FGFR‐independent manner: They regulate neuronal excitability through voltage‐gated sodium channels and bind to intracellular scaffold proteins to mediate unique biological functions. Mechanistically, SPRY proteins inhibit FRS2–GRB2/SOS‐mediated RAS–MAPK signaling; SEF attenuates FGFR‐proximal and MAPK pathway activation; and FGFRL1 acts as a kinase‐deficient FGFR‐like modulator that can sequester ligands or receptor complexes to limit pathway output. Created with Biorender.com. FGF, fibroblast growth factor; FRS2, fibroblast growth factor receptor substrate 2; GRB2, growth factor receptor‐bound protein 2; HSPG, heparan sulfate proteoglycan; JAK, Janus kinase; MAPK, mitogen‐activated protein kinase; PI3K, phosphatidylinositol 3‐kinase; PLCγ, phospholipase C gamma; RAS, rat sarcoma; SOS, Son of Sevenless; STAT, signal transducer and activator of transcription.

Upon activation, FGFRs first phosphorylate the fibroblast growth factor receptor substrate 2 (FRS2). Through its phosphotyrosine‐binding (PTB) domain, FRS2α subsequently recruits the growth factor receptor‐bound protein 2 (GRB2)‐Son of Sevenless (SOS) complex, the phosphatase Src homology region 2 domain‐containing phosphatase‐2 (SHP2), and the adaptor protein GRB2‐associated binding protein 1 (GAB1), leading to the activation of two major downstream cascades: the RAS–MAPK (ERK1/2) pathway and the PI3K–AKT–mammalian target of rapamycin (mTOR) pathway [48, 49, 50]. The MAPK pathway primarily regulates gene expression and the cell cycle, whereas the PI3K pathway maintains cell survival and metabolic homeostasis [51]. FGFRs can directly activate PLCγ, which catalyzes the generation of second messengers, induces calcium ion release from the endoplasmic reticulum (ER) and activates the PKC pathway. This cascade modulates key cellular processes, including cell movement, axon guidance, and cytoskeletal remodeling [52]. Additionally, FGFR‐mediated signaling enhances cell adhesion and participates in tissue morphogenesis through interaction with Src family kinases and the Crk/p130Cas pathway. Under stress conditions, FGFR also regulates the balance between cell survival and death by activating the JNK and p38 MAPK pathways [53].

The cascade of signaling events initiated by FGF ligands ultimately converges upon the cell nucleus, where it precisely modulates the expression of downstream target genes through the action of multiple transcription factors, including STATs, activator protein‐1 (AP‐1), and E26 transformation‐specific (ETS). For instance, FGF/FGFR signaling effectively activates the STAT transcriptional pathway (mainly STAT1, STAT3, and STAT5). This activation can be achieved either by direct phosphorylation of STAT tyrosine residues by the kinase domain of FGFR [54, 55] or indirectly amplifying the STAT activation signals via Src family kinases [54]. Once activated, STAT molecules form homodimers or heterodimers and rapidly translocate to the nucleus, where they function as a transcription factor by binding to the promoters of specific target genes, such as BCL2 and Cyclin D1 [27]. In the context of metabolic regulation, FGF21 exerts atypical effects by activating the AMPKα pathway, thereby regulating energy balance and lipid metabolism [56, 57, 58].

Additionally, FGF19, secreted by the small intestine, reaches the liver via the portal circulation and binds to the FGFR4/β‐Klotho receptor complex on the surface of hepatocytes. This interaction leads to suppression of cholesterol 7α‐hydroxylase (CYP7A1) expression through the activation of the MAPK/ERK/c‐JNK signaling cascade, ultimately reducing BA synthesis [59]. Additionally, FGF23 binds to the FGFR–Klotho complex in the kidney, initiating the MAPK signaling pathway and promoting the internalization of the sodium‐phosphate cotransporters NPT2a and NPT2c on the apical membrane of the proximal epithelial tubules, thereby increasing urinary phosphate excretion [60].

The downstream signaling cascade of FGF/FGFR signaling is governed by stringent, multi‐layered regulatory mechanisms. At the extracellular level, signal amplitude is modulated by the local concentration of FGF ligands, the expression level and spatial distribution of FGFRs, and the extracellular availability of HSPGs [27, 61]. At the intracellular level, negative feedback regulators, including the protein tyrosine phosphatase SHP2 and the lipid phosphatase PTEN, attenuate pathway activity by dephosphorylating key signaling intermediates, thereby preserving signaling homeostasis and preventing hyperactivation [5, 52, 62]. Dysregulation of these control mechanisms, arising from oncogenic FGF or FGFR gene fusions, amplifications, or epigenetic silencing of feedback inhibitors, can result in constitutive pathway activation. Such aberrant signaling contributes to tumorigenesis or monogenic developmental disorders [63, 64]. In addition, Sprouty (SPRY) proteins restrain RAS–MAPK activation by interfering with FRS2‐GRB2/SOS complex formation [65], and similar expression to fgf genes (SEF) serves as a feedback‐induced antagonist of Ras/MAPK pathway‐mediated FGF/FGFR signaling [66]. Moreover, FGFRL1, a kinase‐deficient FGFR‐like receptor, functions as a decoy or modulatory receptor to dampen excessive FGF/FGFR signaling [67].

In summary, the FGF/FGFR signaling is an evolutionarily highly conserved and functionally complex signaling network. Simply put, it acts like a sophisticated “communication system,” transmitting extracellular signals into the cell through the coordinated action of ligands, receptors, co‐receptors, and accessory factors, thereby precisely regulating cellular functions and physiological activities.